Summary: This lab investigates immune responses induced by viral and plasmid vectors. Immune responses can alter the safety and efficacy of gene therapy using viral or plasmid vectors by blocking repeat administration or by causing immunopathology. In addition, results are relevant to vaccine potency and mechanisms of action. Understanding of such immune responses can help us select predictors of clinical success as well as of adverse events, and thus contribute to improved regulatory decision-making. We study the mouse influenza system with special focus on heterosubtypic immunity, that is, immunity induced by influenza A virus of one subtype and providing cross-protection against challenge with flu A of different subtypes. Vaccine development and prevention of pandemics would be aided by a more complete understanding of the broad cross-protection against widely divergent viral strains that is observed in animals. Main projects: a) Immune responses induced by plasmid DNA: DNA vaccines encoding conserved influenza virus antigens reduce challenge virus replication and the resulting morbidity and mortality. We had shown previously that vaccination with DNA encoding influenza nucleoprotein and matrix (NP+M) antigens protected animals, and that either CD4+ or CD8+ T cells could function without the other subset to protect the animal. We have preprepared new constructs encoding other conserved influenza virus components or with features intended to increase expression of the inserted genes and thus induce more potent immunity. We are currently using these constructs along with recombinant viruses in a variety of prime and boost regimens, to investigate protection against challenge and also patterns of epitope dominance. To our previous techniques of antibody analysis and cytotoxic T lymphocyte analysis we have added intracytoplasmic staining for interferon-gamma and TNF-alpha produced by CD4+ or CD8+ T cells. Tetramer staining will also be used to characterize the T cell responses. b) Potential pandemic subtypes: We studied vaccination with DNA encoding the conserved antigens NP and M of influenza virus A/PR/8 (H1N1), followed by challenge with H5N1 viruses from the 1997 outbreak in Hong Kong. H5N1 viruses of low, moderate, and high virulence were used. NP+M DNA vaccination reduced lung virus titers of low and moderate virulence strains, and protected against lethal challenge with the moderate virulence strain. For high virulence virus, the vaccination protected partially against lethal challenge, but could be overwhelmed by high challenge doses. Thus, in the absence of an antigenically-matched HA-based vaccine, this approach may be a useful first line of defense against a rapidly spreading pandemic virus. c) Role of IgA: The role of local, mucosal immune responses in protection against influenza, and in particular in heterosubtypic immunity, is incompletely understood. We have performed mucosal immunizations and heterosubtypic challenges in knockout mice lacking IgA, to analyze the role of this specialized mucosal antibody. Results showed these mice were capable of responses providing some cross-protection of both the upper and lower respiratory tract. However, under some conditions their handling of a primary infection and their protective immunity against heterosubtypic virus differed from that of wild type mice. d) In collaboration with the lab of Dr. Gary Nabel, we are investigating influenza vaccination by a prime-and-boost strategy using recombinant adenoviral vectors. e) Human heterosubtypic immunity: The possible impact of heterosubtypic immunity in humans was investigated by study of archival data from the Cleveland family study performed in the 1940's and 1950's. The 1957 pandemic with its shift from H1N1 to H2N2 virus provided a unique opportunity to analyze heterosubtypic immunity, because the entire human population was naive to H2 and N2 antigens. With IRB approval, records from that study were examined in Cleveland. The data suggest that adults were protected by a form of immunity children did not have.